Plasminogen activators are a unique class of enzymes that convert plasminogen to its active enzymatic form, plasmin. Plasmin is a serine protease that degrades the fibrin network of blood clots. Several plasminogen activators are currently being used as in vivo fibrinolytic agents in the treatment of acute myocardial infarction (Tienfenbrunn et al., 1989, Fibrinolysis 3:1-15). One of these plasminogen activators, tissue plasminogen activator (t-PA), has a significantly enhanced ability to activate plasminogen in the presence of fibrin (Hoylaerts et al., 1982, J. Biol. Chem. 257:2912-2919). Thus, when used as an in vivo fibrinolytic agent, t-PA is directed to fibrin clots.
Human t-PA is a multi-domain serine protease secreted by vascular endothelial cells. The DNA and amino acid structure of human t-PA was described by Pennica et al., 1983, Nature 301:214. Based on the cDNA sequence of t-PA, the inferred sequence of 527 amino acids comprises five distinct structural domains. Gene mapping studies of genomic t-PA have shown that the gene encoding t-PA is comprised of twelve exons split by introns (Ny et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:5355). These introns correspond, in part, with the domain junctions at the amino acid level.
When synthesized by the cell, t-PA comprises a 35 amino acid signal peptide/propeptide region. The signal peptide directs secretion of the t-PA molecule from the cell. The signal peptide/propeptide region is cleaved from the t-PA molecule to yield the 527 amino acid t-PA molecule.
The amino-terminal portion of the t-PA molecule contains a disulfide-linked loop referred to as the Finger domain (F). The second domain, called the Growth Factor domain (E or GF), is highly homologous with epidermal growth factor. The third and fourth domains are highly disulfide-linked structures referred to as Kringles (K1 and K2). The fifth domain, located at the carboxy-terminus, is the Serine Protease domain (SP).
There has been significant growth in the acceptance of thrombolytic therapy for the early restoration of blood flow to ischemic myocardium. However, because of a short plasma half-life, tissue plasminogen activator must be administered by intravenous infusion to insure thrombolytic efficacy. Recent reviews of coronary thrombolysis suggest that the ideal properties of a new thrombolytic agent are that it be fibrin specific, have a longer plasma half-life allowing for a single injection administration, produce more rapid reperfusion, prevent reocclusion and be safer (i.e. lower bleeding risk, non-immunogenic), than streptokinase, t-PA or urokinase (Collen, D., 1988, Klin. Wochenschr. 66 (Suppl. 12):15-23; Bang et al., 1989, Annu. Rev. Pharmacol. Toxicol. 29:322-341; Minno et al., 1989, Pharmacol. Res. 21(2):153-161; Mueller et al., 1989, Med. Clin. North Am. 73(2):387-407).
Since the introduction of recombinant human t-PA, investigators have produced modified recombinant derivatives of t-PA (Van Zonneveld, et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:4670-4674; Krause, J., 1988, Fibrinolysis 2:133-142; Pannekoek et al., 1988, Fibrinolysis 2:123-133; Haigwood et al., 1989, Protein Engineering 2:611-620; Higgins and Bennett, 1990, Annu. Rev. Pharmacol. Toxicol. 30:91). A major emphasis in the design of derivative forms has been to increase fibrin specificity, increase circulating plasma half-life, and decrease bleeding liability compared with the t-PA molecule.
One derivative of t-PA was described by Burck et al., 1990, Journal of Biological Chemistry 265(9):5176. This derivative, known as mt-PA6, was constructed by site-specific mutagenesis of the cDNA encoding human t-PA. The DNA encoding amino acids 4-175 was deleted so that upon expression the resultant t-PA derivative sequentially comprised the signal peptide and propeptide regions of t-PA, the first three amino acids of t-PA, the Kringle 2 domain and the Serine Protease domain. Upon secretion from a eucaryotic host cell, the signal peptide and propeptide regions are cleaved from the molecule. mt-PA6 was found to possess greater fibrinolytic activity and a greater ability to activate thrombus-bound plasminogen than t-PA (Jackson et al., 1990, Circulation 82:930-940). The DNA sequence of mt-PA6 is presented as SEQ. ID. NO: 1. The amino acid sequence of mt-PA6 is presented as SEQ. ID. NO: 2. The DNA encoding the signal peptide and propeptide regions is homologous to that of t-PA and is positioned directly adjacent to (in the 5' direction) the DNA encoding the t-PA molecule. This DNA sequence is presented as SEQ. ID. NO: 9. The amino acid sequence of the signal peptide and propeptide regions is presented as SEQ. ID. NO: 10.
Human t-PA comprises four consensus Asparagine-linked (N-linked) glycosylation sites at amino acids 117, 184, 218 and 448. N-linked glycosylation sites are tripeptide sequences that are specifically recognized and glycosylated. Examples of tripeptide consensus sequences include asparagine-X-serine and asparagine-X-threonine. In t-PA, amino acid 184 in the Kringle 2 domain is glycosylated only part of the time while amino acid 218 is not glycosylated (Higgins and Bennett, supra.). Thus, two glycosylation forms of t-PA have been isolated. One form is glycosylated at amino acid 117 in the Kringle I domain and amino acid 448 in the Serine Protease domain. The second form is glycosylated at amino acids 117, 184 and 448. The oligosaccharides at amino acids 184 and 448 are complex oligosaccharides, while amino acid 117 is occupied by a high-mannose oligosaccharide (Spellman et al., 1989, J. of Biol. Chem. 264(24):14100).
When produced by eucaryotic cell culture, mt-PA6 is also found in two glycosylation forms. These two forms account for the doublet of 40 and 42 kD bands seen when the purified enzyme is analyzed by gel electrophoresis. Primary mt-PA6 (mt-PA6-P) is glycosylated at amino acid 276 (equivalent to amino acid 448 of t-PA) but not at amino acid 12 (equivalent to amino acid 184 of t-PA). Variant mt-PA6 (mt-PA6-V) is glycosylated at amino acids 276 and 12. mt-PA6 lacks the K1 domain and, therefore, lacks the glycosylation site present in this domain. As with t-PA, no glycosylation occurs at amino acid 46 (equivalent to amino acid 218 of t-PA) in the Kringle 2 domain. mt-PA6-V comprises 15-25% of the mt-PA6 molecules secreted from the Syrian hamster cell line AV12-664 (Burck et al., supra).
The thrombolytic potential of mt-PA6-V was studied in a canine thrombosis model. The canine thrombosis model was described by Jackson et al., supra. The role of mt-PA6-V in fibrinogen degradation and plasminogen degradation was also examined by methods described by Jackson et al., supra. These studies demonstrated that diglycosylated t-PA derivatives lacking the Finger, GF and Kringle 1 domains, such as mt-PA6-V, provide advantages over their partially and nonglycosylated counterparts in the treatment of thromboembolic disorders. mt-PA6-V displays the unexpected and advantageous property of causing less systemic conversion of plasminogen to plasmin. mt-PA6-V is markedly less prone to metabolism to its two-chain form, and has a higher fibrinolytic versus fibrinogenolytic ratio than mt-PA6-P. Surprisingly, mt-PA6-V also provides a greater maintenance of coronary blood flow. Methods for the use of diglycosylated forms of t-PA derivatives that lack the Finger, Growth Factor and Kringle 1 domains in the treatment of thromboembolic disorders are disclosed by U.S. patent application Ser. No. 07/633,584, filed Dec. 24, 1990, U.S. Pat. No. 5,242,688.
As noted above, glycosylation at amino acid 184 of t-PA, or the equivalent position in other t-PA derivatives such as mt-PA6, occurs in only a small portion of the molecules. For example, mt-PA6-V comprises only 15%-25% Of the mt-PA6 molecules secreted from a Syrian Hamster cell line (Burck et al., supra.). In view of the beneficial properties of diglycosylated forms of t-PA derivatives that lack the Finger, Growth Factor and Kringle 1 domains, it would be advantageous to be able to produce a homogeneous population of these diglycosylated t-PA derivative from a host cell.
The present invention addresses this need by providing novel diglycosylated t-PA derivatives. The invention further provides DNA compounds, expression vectors and transformed host cell which enable the production of a homogeneous population of these derivatives. The invention further provides methods and compositions for the treatment of thromboembolic disorders.
For purposes of the present invention, as disclosed and claimed herein, the following terms are defined below.
ApR--the ampicillin-resistant phenotype or gene conferring same.
E1A--an immediate-early gene product of adenovirus which can activate a poly-GT element to express enhancer activity and can activate the BK virus enhancer.
ep--a DNA segment comprising the SV40 early promoter of the T-antigen gene, the T-antigen binding sites, and the SV40 origin of replication.
GBMT transcription control unit--a modified transcription control unit that comprises the P2 enhancer element of BK virus spaced closely to the upstream regulatory element of the major late promoter of adenovirus (MLTF), the adenovirus-2 major late promoter and a poly-GT element positioned to stimulate said promoter and a DNA sequence encoding the spliced tripartite leader of adenovirus-2. The GMBT transcription control unit is best exemplified by the approximately 900 base pair HindIII cassette found in plasmid pGTC which is found in Escherichia coli K-12 AG1/pGTC (NRRL B-18593).
GT--enhancer system--any poly-GT element linked to a promoter, such as MLP, in which the poly-GT element does not itself possess enhancer activity but is activated as an enhancer by an immediate-early gene product of a large DNA virus, such as the E1A gene product or by any similarly activating viral gene product.
HmR--the hygromycin-resistant phenotype or gene conferring same.
IVS--DNA encoding an intron, also called an intervening sequence.
Large DNA virus--a virus that infects eucaryotic cells and has a genome greater than .about.10 kb in size, i.e., any of the pox viruses, adenoviruses, and herpes viruses.
MLP--the major late promoter of adenovirus, that is also referred to herein as the adenovirus late promoter, adenovirus-type-2 late promoter, or Ad2 late promoter.
MLTF binding site--the site in adenovirus DNA where the major late transcription factor (MLTF) binds; the MLTF is required for MLP activity.
Glycosylation--the attachment of oligosaccharides to a protein through an N-glycosidic bond with the asparagine residue in an Ash-X-Ser/Thr sequence.
NeoR--the neomycin resistance-conferring gene, which can also be used to confer G418 resistance in eucaryotic host cells.
ori--a plasmid origin of replication.
pA--a DNA sequence encoding a polyadenylation signal.
Poly-GT element--a DNA sequence of (GT)n-(CA)n, which is illustrated herein by a sequence where n is 21, but which can also refer to sequences of varying lengths where n is greater or less than 21, and may refer to chemically synthesized (GT)n-(CA)n sequences or human genomic DNA fragments containing a (GT)n-(CA)n tract.
Reocclusion--complete cessation of blood flow after successful thrombolysis caused by reformation of thrombus and/or vasoconstriction.
Reperfusion--restoration of blood flow caused by successful thrombolysis.